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SA- β-Gal assay was performed using cellular senescence assay kit as the manufacturer's instruction (Cell Signaling).
According to manufacturer's instruction, cell invasion and migration assays were performed using a transwell membrane (Corning Costar Corporation, Cambridge, MA, USA).
Protein carbonyls were measured using an immunoblot kit to detect the DNPH derivatization of protein carbonyls, as per the manufacturer's instruction (Cell Biolabs, San Diego, CA, USA).
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According to the manufacturer's instruction cells were washed with a buffer solution containing sodium citrate, sucrose, and dimethyl sulfoxide (DMSO).
However, duck red blood cells were used as instruction cells, and NDV JSD0812 strain homologous to that used for inoculation was used as antigen in HI tests.
Phospho-p38 was measured using an ELISA kit according to the manufacturer's instructions (Cell Signaling, Beverly, MA).
Titration of recombinant adenovirus was performed by a plaque forming unit (PFU) assay in 293 cells following the manufacturer's instructions (Cell Biolabs).
For CREB knockdown, U251 and 5310 cells were transfected with CREB siRNA for 48 hrs using FuGene according to manufacturer's instructions (Cell Signaling Tech., Danvers, MA).
Cell viability was then measured using a fluorometric assay according to the manufacturer's instructions (Cell Titer Blue assay; Promega, Madison, WI) and expressed as percentage of the value measured by cells treated with the negative control.
Recombinant protein expression was detected by Western blot of transfected cell lysates or by immunocytochemistry on fixed (not permeabilized) cells using HA-Tag (6E2) mouse monoclonal antibodies according to the manufacturer's instructions (Cell Signaling).
Immunofluorescence was performed according to the manufacturer's instructions (Cell signaling).
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